Phloretin differentially inhibits volume-sensitive and cyclic AMP-activated, but not Ca-activated, Cl(-) channels.
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ABSTRACT:
SUBMITTER: Fan HT
PROVIDER: S-EPMC1572865 | biostudies-other | 2001 Aug
REPOSITORIES: biostudies-other
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Project description:To ensure specificity of response, eukaryotic cells often restrict signalling molecules to sub-cellular regions. The Ca2+ nanodomain is a spatially confined signal that arises near open Ca2+ channels. Ca2+ nanodomains near store-operated Orai1 channels stimulate the protein phosphatase calcineurin, which activates the transcription factor NFAT1, and both enzyme and target are initially attached to the plasma membrane through the scaffolding protein AKAP79. Here, we show that a cAMP signalling nexus also forms adjacent to Orai1. Protein kinase A and phosphodiesterase 4, an enzyme that rapidly breaks down cAMP, both associate with AKAP79 and realign close to Orai1 after stimulation. PCR and mass spectrometry failed to show expression of Ca2+-activated adenylyl cyclase 8 in HEK293 cells, whereas the enzyme was observed in neuronal cell lines. FRET and biochemical measurements of bulk cAMP and protein kinase A activity consistently failed to show an increase in adenylyl cyclase activity following even a large rise in cytosolic Ca2+. Furthermore, expression of AKAP79-CUTie, a cAMP FRET sensor tethered to AKAP79, did not report a rise in cAMP after stimulation, despite AKAP79 association with Orai1. Hence, HEK293 cells do not express functional active Ca2+-activated adenylyl cyclases including adenylyl cyclase 8. Our results show that two ancient second messengers are independently generated in nanodomains close to Orai1 Ca2+ channels.
| S-EPMC8394516 | biostudies-literature
Project description:All vertebrate cells activate Cl- currents (ICl ,swell) when swollen by hypotonic bath solution. The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca2+-activated Cl- channel (CaCC) TMEM16A and the phospholipid scramblase and ion channel TMEM16F were suggested to contribute to cell swelling-activated whole-cell currents. Cell swelling was shown to induce Ca2+ release from the endoplasmic reticulum and to cause subsequent Ca2+ influx. It is suggested that TMEM16A/F support intracellular Ca2+ signaling and thus Ca2+-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to ICl ,swell. In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of ICl ,swell by hypotonic bath solution (Hypo; 200 mosm/l) was Ca2+ dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca2+ concentrations. In HT29 cells, knockdown of endogenous TMEM16A attenuated ICl ,swell and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of ICl ,swell by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine (IP3R; RyR), as well as by inhibiting Ca2+ influx. The data suggest that TMEM16A contributes directly to ICl ,swell as it is activated through swelling-induced Ca2+ increase. As activation of VRAC is shown to be Ca2+-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca2+ increase in submembraneous signaling compartments by means of ER tethering.
| S-EPMC7736618 | biostudies-literature
Project description:BackgroundTransmembrane protein 16A (TMEM16A) Ca2+-activated Cl- channels have diverse physiological functions, such as epithelial secretion of Cl- and fluid and sensation of pain. Recent studies have demonstrated that TMEM16A contributes to the pathogenesis of infectious and non-infectious inflammatory diseases. However, the role of TMEM16A in inflammation has not been clearly elucidated.Aim of reviewIn this review, we aimed to provide comprehensive information regarding the roles of TMEM16A in inflammation by summarizing the mechanisms underlying TMEM16A expression and activation under inflammatory conditions, in addition to exploring the diverse inflammatory signaling pathways activated by TMEM16A. This review attempts to develop the idea that TMEM16A plays a diverse role in inflammatory processes and contributes to inflammatory diseases in a cellular environment-dependent manner.Key scientific concepts of reviewMultiple inflammatory mediators, including cytokines (e.g., interleukin (IL)-4, IL-13, IL-6), histamine, bradykinin, and ATP/UTP, as well as bacterial and viral infections, promote TMEM16A expression and/or activity under inflammatory conditions. In addition, TMEM16A activates diverse inflammatory signaling pathways, including the IP3R-mediated Ca2+ signaling pathway, the NF-κB signaling pathway, and the ERK signaling pathway, and contributes to the pathogenesis of many inflammatory diseases. These diseases include airway inflammatory diseases, lipopolysaccharide-induced intestinal epithelial barrier dysfunction, acute pancreatitis, and steatohepatitis. TMEM16A also plays multiple roles in inflammatory processes by increasing vascular permeability and leukocyte adhesion, promoting inflammatory cytokine release, and sensing inflammation-induced pain. Furthermore, TMEM16A plays its diverse pathological roles in different inflammatory diseases depending on the disease severity, proliferating status of the cells, and its interacting partners. We herein propose cellular environment-dependent mechanisms that explain the diverse roles of TMEM16A in inflammation.
| S-EPMC8463915 | biostudies-literature
Project description:Ca2+-activated Cl- channels (CaCCs) perform a multitude of functions including the control of cell excitability, regulation of cell volume and ionic homeostasis, exocrine and endocrine secretion, fertilization, amplification of olfactory sensory function, and control of smooth muscle cell contractility. CaCCs are the translated products of two members (ANO1 and ANO2, also known as TMEM16A and TMEM16B) of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca2+, post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca2+ or indirectly through the intervention of the Ca2+-binding protein Calmodulin (CaM), and the structural domains responsible for Ca2+- and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl-(4,5)-bisphosphate (PIP2), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ+ channel β subunits KCNE1/5.
| S-EPMC8480199 | biostudies-literature
Project description:Chloroquine (CQ), a bitter tasting drug widely used in treatment of malaria, is associated gastrointestinal side effects including nausea or diarrhea. In the present study, we investigated the effect of CQ on electrolyte transport in rat ileum using the Ussing chamber technique. The results showed that CQ evoked an increase in short circuit current (ISC) in rat ileum at lower concentration (?5×10(-4) M) but induced a decrease at higher concentrations (?10(-3) M). These responses were not affected by tetrodotoxin (TTX). Other bitter compounds, such as denatoniumbenzoate and quinine, exhibited similar effects. CQ-evoked increase in ISC was partly reduced by amiloride(10(-4) M), a blocker of epithelial Na(+) channels. Furosemide (10(-4) M), an inhibitor of Na(+)-K(+)-2Cl(-) co-transporter, also inhibited the increased ISC response to CQ, whereas another Cl(-) channel inhibitor, CFTR(inh)-172(10(-5) M), had no effect. Intriguingly, CQ-evoked increases were almost completely abolished by niflumic acid (10(-4) M), a relatively specific Ca(2+)-activated Cl(-) channel (CaCC) inhibitor. Furthermore, other CaCC inhibitors, such as DIDS and NPPB, also exhibited similar effects. CQ-induced increases in ISC were also abolished by thapsigargin(10(-6) M), a Ca(2+) pump inhibitor and in the absence of either Cl(-) or Ca(2+) from bathing solutions. Further studies demonstrated that T2R and CaCC-TMEM16A were colocalized in small intestinal epithelial cells and the T2R agonist CQ evoked an increase of intracelluar Ca(2+) in small intestinal epithelial cells. Taken together, these results demonstrate that CQ induces Cl(-) secretion in rat ileum through CaCC at low concentrations, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.
| S-EPMC3907514 | biostudies-literature
Project description:IntroductionTransmembrane protein 16A (TMEM16A) is a Ca2+-activated chloride channel that plays a role in cancer cell proliferation, migration, invasion, and metastasis. However, whether TMEM16A contributes to breast cancer metastasis remains unknown.ObjectiveIn this study, we investigated whether TMEM16A channel activation by ROCK1/moesin promotes breast cancer metastasis.MethodsWound healing assays and transwell migration and invasion assays were performed to study the migration and invasion of MCF-7 and T47D breast cancer cells. Western blotting was performed to evaluate the protein expression, and whole-cell patch clamp recordings were used to record TMEM16A Cl- currents. A mouse model of breast cancer lung metastasis was generated by injecting MCF-7 cells via the tail vein. Metastatic nodules in the lung were assessed by hematoxylin and eosin staining. Lymph node metastasis, overall survival, and metastasis-free survival of breast cancer patients were assessed using immunohistochemistry and The Cancer Genome Atlas dataset.ResultsTMEM16A activation promoted breast cancer cell migration and invasion in vitro as well as breast cancer metastasis in mice. Patients with breast cancer who had higher TMEM16A levels showed greater lymph node metastasis and shorter survival. Mechanistically, TMEM16A promoted migration and invasion by activating EGFR/STAT3/ROCK1 signaling, and the role of the TMEM16A channel activity was important in this respect. ROCK1 activation by RhoA enhanced the TMEM16A channel activity via the phosphorylation of moesin at T558. The cooperative action of TMEM16A and ROCK1 was supported through clinical findings indicating that breast cancer patients with high levels of TMEM16A/ROCK1 expression showed greater lymph node metastasis and poor survival.ConclusionOur findings revealed a novel mechanism underlying TMEM16A-mediated breast cancer metastasis, in which ROCK1 increased TMEM16A channel activity via moesin phosphorylation and the increase in TMEM16A channel activities promoted cell migration and invasion. TMEM16A inhibition may be a novel strategy for treating breast cancer metastasis.
| S-EPMC8463928 | biostudies-literature
Project description:Ca(2+)-activated Cl(-) channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca(2+) sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca(2+)-dependent Cl(-) transport with submicromolar sensitivity to Ca(2+), consistent with what is seen in patch-clamp experiments. The channel is synergistically gated by Ca(2+) and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca(2+) dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl(-) transport with Kis comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.
| S-EPMC3845129 | biostudies-literature
Project description:BACKGROUND AND PURPOSE: The protoberberine alkaloid berberine has been reported to inhibit colonic Cl(-) secretion. However, it is not known if other protoberberine alkaloids share these effects. We have therefore selected another protoberberine alkaloid, palmatine, to assess its effects on active ion transport across rat colonic epithelium. EXPERIMENTAL APPROACH: Rat colonic mucosa was mounted in Ussing chambers and short circuit current (I (SC)), apical Cl(-) current and basolateral K(+) current were recorded. Intracellular cAMP content was determined by an enzyme immunoassay. Intracellular Ca(2+) concentration was measured with Fura-2 AM. KEY RESULTS: Palmatine inhibited carbachol-induced Ca(2+)-activated Cl(-) secretion and the carbachol-induced increase of intracellular Ca(2+) concentration. Palmatine also inhibited cAMP-activated Cl(-) secretion induced by prostaglandin E(2) (PGE(2)) or forskolin. Palmatine prevented the elevation of intracellular cAMP by forskolin. Determination of apical Cl(-) currents showed that palmatine suppressed the forskolin-stimulated, apical cAMP-activated Cl(-) current but not the carbachol-stimulated apical Ca(2+)-activated Cl(-) current. Following permeabilization of apical membranes with nystatin, we found that palmatine inhibited a carbachol-stimulated basolateral K(+) current that was sensitive to charybdotoxin and resistant to chromanol 293B. However, the forskolin-stimulated basolateral K(+) current inhibited by palmatine was specifically blocked by chromanol 293B and not by charybdotoxin. CONCLUSIONS AND IMPLICATIONS: Palmatine attenuated Ca(2+)-activated Cl(-) secretion through inhibiting basolateral charybdotoxin-sensitive, SK4 K(+) channels, whereas it inhibited cAMP-activated Cl(-) secretion by inhibiting apical CFTR Cl(-) channels and basolateral chromanol 293B-sensitive, KvLQT1 K(+) channels.
| S-EPMC2275457 | biostudies-other
Project description:The KCNQ1 K(+) channel plays a key role in the regulation of several physiological functions, including cardiac excitability, cardiovascular tone, and body electrolyte homeostasis. The metabolic sensor AMP-activated protein kinase (AMPK) has been shown to regulate a growing number of ion transport proteins. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus laevis oocytes and collecting duct epithelial cells. AMPK activation decreased KCNQ1 currents and channel surface expression in X. laevis oocytes, but AMPK did not phosphorylate KCNQ1 in vitro, suggesting an indirect regulatory mechanism. As it has been recently shown that the ubiquitin-protein ligase Nedd4-2 inhibits KCNQ1 plasma membrane expression and that AMPK regulates epithelial Na(+) channels via Nedd4-2, we examined the role of Nedd4-2 in the AMPK-dependent regulation of KCNQ1. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant-negative or constitutively active Nedd4-2 mutant, or a Nedd4-2 interaction-deficient KCNQ1 mutant, suggesting that Nedd4-2 participates in the regulation of KCNQ1 by AMPK. KCNQ1 is expressed at the basolateral membrane in mouse polarized kidney cortical collecting duct (mpkCCD(c14)) cells and in rat kidney. Treatment with the AMPK activators AICAR (2 mM) or metformin (1 mM) reduced basolateral KCNQ1 currents in apically permeabilized polarized mpkCCD(c14) cells. Moreover, AICAR treatment of rat kidney slices ex vivo induced AMPK activation and intracellular redistribution of KCNQ1 from the basolateral membrane in collecting duct principal cells. AICAR treatment also induced increased ubiquitination of KCNQ1 immunoprecipitated from kidney slice homogenates. These results indicate that AMPK inhibits KCNQ1 activity by promoting Nedd4-2-dependent channel ubiquitination and retrieval from the plasma membrane.
| S-EPMC3006313 | biostudies-literature
Project description:In the olfactory bulb, input from olfactory receptor neurons is processed by neuronal networks before it is relayed to higher brain regions. In many neurons, hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels generate and control oscillations of the membrane potential. Oscillations also appear crucial for information processing in the olfactory bulb. Four channel isoforms exist (HCN1-HCN4) that can form homo- or heteromers. Here, we describe the expression pattern of HCN isoforms in the olfactory bulb of mice by using a novel and comprehensive set of antibodies against all four isoforms. HCN isoforms are abundantly expressed in the olfactory bulb. HCN channels can be detected in most cell populations identified by commonly used marker antibodies. The combination of staining with marker and HCN antibodies has revealed at least 17 different staining patterns in juxtaglomerular cells. Furthermore, HCN isoforms give rise to an unexpected wealth of co-expression patterns but are rarely expressed in the same combination and at the same level in two given cell populations. Therefore, heteromeric HCN channels may exist in several cell populations in vivo. Our results suggest that HCN channels play an important role in olfactory information processing. The staining patterns are consistent with the possibility that both homomeric and heteromeric HCN channels are involved in oscillations of the membrane potential of juxtaglomerular cells.
| S-EPMC2838509 | biostudies-literature